Bead-based analysis of a sample

ABSTRACT

A method includes attaching two or more beads to each unit of one or more units of a chemical component in a sample, to form, for each unit of the chemical component, a multi-bead complex including two or more beads and the unit of the chemical component; placing the sample on a surface of an image sensor; at the image sensor, receiving light originating at a light source, the received light including light reflected by, refracted by, or transmitted through the beads of the multi-bead complexes; at the image sensor, capturing one or more images of the sample from the received light; and identifying, in at least one of the images of the sample, separate multi-bead complexes, the identifying of the separate multi-bead complexes including associating the two or more beads of each of the multi-bead complexes based on proximity to one another.

PRIORITY CLAIMS

This application is a divisional of U.S. Pat. Application 17/674,967,filed on Feb. 18, 2022, which is a divisional of U.S. Pat. Application16/845,458, filed on Apr. 10, 2020 (U.S. Pat. No. 11,255,850, whichissued on Feb. 22, 2022), which is a continuation-in-part of U.S. Pat.Application 16/368,707, filed on Mar. 28, 2019 (U.S. Pat. No.10,684,278, which issued on Jun. 16, 2020), the entire contents of eachof which are incorporated here by reference.

BACKGROUND

This description relates to bead-based analysis of a sample.

To obtain all the useful information in a sample of whole blood of apatient for purposes of diagnosis, for example, requires not only acomplete blood count (CBC) of the various types of blood cells in theblood sample and their hemoglobin content but also a chemical analysisof other components in the acellular portion of blood (e.g., theplasma). Such other components can include molecules and ions of variouskinds.

Traditionally, both a CBC and a chemical analysis of blood are performedin a lab on large expensive machines using tubes of venous bloodobtained by phlebotomy. Hours or days may be required for the chemicalanalysis to be completed and the results returned.

SUMMARY

In general, an aspect of the disclosure is a method including attachingtwo or more beads to each unit of one or more units of a chemicalcomponent in a sample, to form, for each unit of the chemical component,a multi-bead complex including two or more beads and the unit of thechemical component; placing the sample on a surface of an image sensor;at the image sensor, receiving light originating at a light source, thereceived light including light reflected by, refracted by, ortransmitted through the beads of the multi-bead complexes; at the imagesensor, capturing one or more images of the sample from the receivedlight; and identifying, in at least one of the images of the sample,separate multi-bead complexes, the identifying of the separatemulti-bead complexes including associating the two or more beads of eachof the multi-bead complexes based on proximity to one another.

Implementations may include one or a combination of two or more of thefollowing features. The method includes identifying a presence of thechemical component based on the identification of the separatemulti-bead complexes. The method includes identifying a level of thechemical component based on the identification of the separatemulti-bead complexes. Identifying the separate multi-bead complexesincludes enumerating the separate multi-bead complexes.

In some implementations, attaching two or more beads to each unit of thechemical component includes binding two or more attachment units to eachunit of the one or more units of the chemical component, each of theattachment units also being attached to one or more beads, such thateach multi-bead complex includes two or more beads, two or moreattachment units, and the unit of the chemical component. In someimplementations, the attachment units include antibodies. In someimplementations, the attachment units include capsid proteins or otherantigens from a pathogen, and the units of the chemical componentinclude antibodies to the pathogen. In some implementations, thepathogen includes a virus. In some implementations, at least two of thetwo or more attachment units are different from one another. In someimplementations, the two or more attachment units bind at differentlocations of a unit of the chemical component.

Implementations may include one or a combination of two or more of thefollowing features. At least two of the two or more beads of amulti-bead complex have the same reflective, refractive, andtransmissive characteristics. At least two of the two or more beads of amulti-bead complex have different reflective, refractive, ortransmissive characteristics or combinations of them for the lightoriginating at the light source. The different reflective, refractive,or transmissive characteristics include at least one of colors of thebeads, sizes of the beads, shapes of the beads, and birefringence of thebeads. Each unit of the chemical component includes an antibody of apathogenic virus. Placing the sample on the surface of the image sensorincludes forming a monolayer of the sample on the surface. Placing thesample on the surface of the image sensor includes confining the samplebetween the surface of the image sensor and a second surface oppositethe surface of the image sensor. The method includes identifying, in atleast one of the images of the sample, separate individual beads basedon light reflected by, refracted by, or transmitted by each of theseparate individual beads, and based on a proximity of each of theseparate individual beads to other beads.

In general, an aspect of the disclosure is an apparatus including animage sensor having an array of light sensitive elements at a surface ofthe image sensor, and one or more computing processors communicativelycoupled to the image sensor, the one or more computing processorsconfigured to perform operations including: receiving, from the imagesensor, data representative of one or more images of a sample situatedat the surface of the image sensor, the sample including one or moremulti-bead complexes, each multi-bead complex including two or morebeads attached to a unit of a chemical component, in which the one ormore images are based on light originating at a light source andreceived at the image sensor, the received light including lightreflected by, refracted by, or transmitted through the beads of themulti-bead complexes; and identifying, in at least one of the images ofthe sample, separate multi-bead complexes, the identifying of theseparate multi-bead complexes including associating the two or morebeads of each of the multi-bead complexes based on close proximity toone another.

Implementations may include one or a combination of two or more of thefollowing. The operations include identifying a presence of the chemicalcomponent based on the identification of the separate multi-beadcomplexes. The operations include identifying a level of the chemicalcomponent based on the identification of the separate multi-beadcomplexes. Identifying the separate multi-bead complexes includesenumerating the separate multi-bead complexes.

In some implementations, each multi-bead complex includes two or moreattachment units bound to a unit of the chemical component, each of theattachment units also being attached to one or more beads, such thateach multi-bead complex includes two or more beads, two or moreattachment units, and the unit of the chemical component. In someimplementations, the attachment units include antibodies. In someimplementations, the attachment units include capsid proteins or otherantigens from a pathogen, and the units of the chemical componentinclude antibodies to the pathogen. In some implementations, thepathogen includes a virus. In some implementations, at least two of thetwo or more attachment units are different from one another. In someimplementations, the two or more attachment units bind at differentlocations of a unit of the chemical component.

Implementations may include one or a combination of two or more of thefollowing. At least two of the two or more beads of a multi-bead complexhave the same reflective, refractive, and transmissive characteristics,and the one or more processors are configured to detect the reflective,refractive, and transmissive characteristics. At least two of the two ormore beads of a multi-bead complex have different reflective,refractive, or transmissive characteristics or combinations of them forthe light originating at the light source, and the one or moreprocessors are configured to detect the reflective refractive, andtransmissive characteristics. The different reflective, refractive, ortransmissive characteristics include at least one of colors of thebeads, sizes of the beads, shapes of the beads, and birefringence of thebeads. Each unit of the chemical component includes an antibody of apathogenic virus. The apparatus includes a second surface opposite thesurface of the image sensor, the second surface configured to confinethe sample between the second surface and the surface of the imagesensor. The second surface is configured to form a monolayer of thesample between the second surface and the surface of the image sensor.The apparatus includes a light source. The operations includeenumerating, in at least one of the images of the sample, separateindividual beads based on light reflected by, refracted by, ortransmitted by each of the separate individual beads, and based on aproximity of each of the separate individual beads to other beads.

These and other aspects, features, implementations, and advantages (1)can be expressed as methods, apparatus, systems, components, programproducts, business methods, means or steps for performing functions, andin other ways, and (2) will become apparent from the followingdescription and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of an example fluorescence immunoassaytechnique.

FIG. 2 is a schematic view of an example sample analysis device.

FIG. 3 is a schematic view of an example bead-complexing technique forsample analysis.

FIG. 4 is a graph of a standard curve.

FIG. 5 is a schematic view of an example surface including captureantibodies.

FIG. 6 is a schematic view of an example surface including a boundattachment element.

DETAILED DESCRIPTION

Here we describe a sample analysis technology that in someimplementations can perform a chemical analysis of a sample of wholeblood alone or in combination with a CBC directly at a point of carewithin a few minutes at low cost using a small portable easy-to-use,relatively inexpensive sample analysis device. In some uses, because ofits small size and low cost, the sample analysis device can bereproduced in large numbers and distributed to many locations within oneor more healthcare, residential, industrial, or commercial locations. Insome applications, many units of the sample analysis device can bedistributed and used in the field including at locations where equipmentfor sample analysis (for example, blood chemistry or CBC) is otherwiseunavailable or prohibitively expensive.

We use the term “point-of-care” broadly to include, for example, anylocation in close physical proximity to a patient or other person towhom healthcare is being provided. In many cases, point-of-care refersto services provided in the physical presence of a patient, for example,in the same room or building or at the same place or within a shortdistance.

Although much of the discussion below refers to applications of thesample analysis technology to chemical analysis of whole blood drawnfrom a human or other animal, the sample analysis technology can also beapplied to a wide range of contexts in which a sample (which may, butneed not, be a biological sample) contains chemical components ofinterest (such as molecules or ions) and that may not involve countingand may or may not include particles, units, or other elements of one ormore kinds that are to be counted.

We use the term “sample” broadly to include, for example, any fluid orother mass or body of material that contains one or more analyzablechemical components and may or may not also contain one or morecountable units of one or more types. The countable units may in somecases be opaque, translucent, or otherwise non-transparent to incidentlight. The analyzable chemical components may in some instances betransparent, translucent, or otherwise non-opaque to incident light. Insome examples, the sample is whole blood containing countable bloodcells of different types and also containing analyzable chemicalcomponents such as molecules or ions, to name two.

We use the term “chemical components” broadly to include, for example,chemical compounds, ions, molecules, and other constituents of a samplethat may not be present in a form of discernible (e.g., visible)countable units.

We use the term “unit of a chemical component” broadly to include, forexample, a single unit of a chemical component such as a singlemolecule, ion, or other constituent. In typical samples, there are manyunits of a given type of chemical component, for example, many moleculesof a chemical compound.

We use the term “countable units” broadly to include, for example,elements present in a sample that are discrete, discernible, visible,identifiable, and subject to enumeration. Typically, countable units arenot transparent. In the case of whole blood, the countable units caninclude blood cells of different types.

We use the term “chemical analysis” broadly to include, for example,identification and quantification (e.g., determination of the level) ofchemical components of one or more types in the sample. In some cases,chemical analysis can include identifying the presence of one or moremolecules of one or more types and characterizing the amount, volume, orpercentage of each of the types of molecules in the sample or in aparticular volume of the sample.

As noted earlier, although the sample analysis technology has a broaderrange of applications, for convenience we sometimes discuss particularexamples in which the sample includes whole blood or components of wholeblood.

We use the term “whole blood” broadly to include, for example, blood inits original form drawn from a human or other animal. Whole bloodincludes countable units such as blood cells and blood plasma thatincludes chemical components. As described in the Wikipedia entry titled“Blood plasma” blood plasma is “a yellowish liquid component of bloodthat normally holds the blood cells in whole blood in suspension. Inother words, it is the liquid part of the blood that carries cells andproteins ... It is mostly water (up to 95% by volume), and containsdissolved proteins (6-8%) (e.g. serum albumins, globulins, andfibrinogen), glucose, clotting factors, electrolytes (Na⁺, Ca²⁺, Mg²⁺,HCO₃ ⁻, Cl⁻, etc.), hormones, carbon dioxide (plasma being the mainmedium for excretory product transportation) and oxygen.” Clottingfactors include molecules such as plasminogen and prothrombin thatparticipate in clot formation.

We use the term “blood cells” broadly to include, for example, red bloodcells (erythrocytes), white blood cells (leukocytes), rare blood celltypes, ambiguous blood cell types, and platelets (thrombocytes).

As shown in FIG. 1 , typical automated techniques 10 for chemicalanalysis of blood use fluorescence-based sandwich immunoassay techniquesto identify and quantify acellular chemical components 12, for example,molecules of one or more chemical components in blood plasma 14. The“filling” of the “sandwich” in fluorescence-based sandwich immunoassayis, for example, molecules 16 of a given target chemical component inthe blood plasma. Each of the molecules is, in effect, sandwiched 18 asa result of adding two types 20, 22 of antibodies to the blood sample.The antibodies of one type 20 are known to bind specifically to onelocation 24 on the target molecules and serve as “capture antibodies” inthe sense that they provide a known “base” at which the target moleculesare held. The antibodies of the other type 22 serve as “detectionantibodies” and are also known to bind specifically to the targetmolecules, but to a different location 26 on the target molecules. Insome examples, the capture antibodies are fixed, say, to a surface 28and literally “capture” the target molecules and hold them at aparticular location on the surface. The detection antibodies aretypically marked by fluorescent molecules 30 attached to them.

Once the target molecules have been captured, that is, bound to thecapture antibodies, high intensity excitation light 32 illuminates thesample in one wavelength band causing much lower intensity light to beemitted 34 from the attached fluorescent molecules in a different,typically longer, fluorescence wavelength band. The emitted light issensed by a light detector 36 (after being passed through a filter 38 toblock the much higher intensity excitation light). The light detector ishighly sensitive to the presence and intensity level of the relativelylow intensity fluorescence wavelength band light and can thereforegenerate signals indicating the fluorescence intensity and in turn theamount of the target chemical component present in the sample.

The fluorescence sandwich technique can be used to identify and quantifydifferent target chemical components of blood simultaneously by usingdifferent appropriate pairs of capture antibodies and suitably labelled(by fluorescent molecules) detection antibodies. In some implementationsof such multiplexing, the different capture antibodies are attached atdifferent locations to a fixed surface as a way to differentiate thedifferent target molecules based on their locations at the fixedsurface. In some implementations, the target molecules remain dissolvedor suspended in the sample and the different capture antibodies aremarked using fluorescent beads (for example, Luminex® beads) thatproduce fluorescence light in different wavelength bands, or differentcombinations of the bands, as a way to differentiate the different typesof target molecules without regard to their locations in the sample.

As discussed later, in some implementations of the sample analysistechnology, chemical analysis is combined with a contact monolayernon-fluorescence imaging technique for performing a complete blood count(CBC). For several reasons, the standard fluorescence sandwich techniquejust described is not optimally compatible with the contact monolayernon-fluorescence CBC technique. One reason is that, in the contact CBCtechnique, the blood sample is typically in direct contact with alight-sensitive surface of an image sensor which precludes the inclusionof a filter element between the surface and the sample to block the highintensity excitation light. A second reason is that the contact CBCtechnique is not readily compatible with washing and other processingsteps (one of which involves removing non-transparent blood cells fromthe sample) generally required in fluorescence sandwich immunoassaytechniques. The washing and processing steps cannot be easily applied ifthe same whole blood sample used for the sample analysis technique is tobe used also for the contact CBC technique. [Yet, as will be discussedlater, because the contact CBC technique is based on the use of amonolayer of blood, portions of the monolayer are free of blood cellsand contain only light-passing blood plasma. Therefore, although theentire area of the image sensor may not be suitable for chemicalanalysis of the target molecules because of the presence of blood cells,some portions of the area of the image sensor are suitable for thesample analysis technique even with whole blood.] A third reason why thefluorescent sandwich technique is not optimally compatible with thecontact CBC technique described above is that the small size pixels ofthe high-resolution image sensor do not provide as adequate low-lightsensitivity to detect the low intensity emitted fluorescence light ascan a larger-area light detector.

The sample analysis technology that is described here can be usedindependently to perform chemical analysis of whole blood or can be usedto perform chemical analysis of whole blood in combination with or tosupplement (simultaneously or sequentially) a contact CBC technique thatuses the same sample and light from the same light source. As a result,both the contact CBC technique and the blood chemical analysis can beperformed quickly at essentially the same time on a tiny sample of wholeblood (for example, a sample of less than 50 microliters or less then 15microliters or less then 5 microliters) at the point-of-care using asmall inexpensive device. Although we often discuss examples in whichthe chemical analysis is performed on whole blood, the sample analysistechnology can be applied to raw whole blood or to whole blood that hasbeen processed to alter or adjust or remove or supplement chemicalcomponents or to whole blood from which some or all of the blood cellshave been removed, including blood plasma.

We use the term “contact CBC technique” broadly to include, for example,any technique in which blood cells of one or more types are identifiedand counted in a sample that is in contact with (e.g., within anear-field distance of) a surface of an image sensor. Additionalinformation about contact CBC techniques can be found in one or more ofU.S. Pat. Publications 2016/0041200, 2014/0152801, 2018/0284416,2017/0293133, 2016/0187235, and U.S. Pat. 9,041,790, 9,720,217,10,114,203, 9,075,225, 9,518,920, 9,989,750, 9,910,254, 9,952,417,10,107,997, all of which are incorporated here by reference.

Referring to FIG. 2 , in some implementations of the sample analysistechnology, a monolayer 100 of whole blood is situated between a surface102 of a high resolution image sensor 104 at which an array ofphotosensitive elements (e.g., pixels) 106 are exposed and acorresponding surface 108 of a lid 110, to form a monolayer having aknown volume defined by its length, width, and thickness 112 between thesurface 102 and the surface 108. Examples of structures and techniquesfor forming such a monolayer are described in one or more of U.S. Pat.Publications 2016/0041200, 2014/0152801, 2018/0284416, 2017/0293133,2016/0187235, and in U.S. Pats. 9,041,790, 9,720,217, 10,114,203,9,075,225, 9,518,920, 9,989,750, 9,910,254, 9,952,417, 10,107,997, allof which are incorporated here by reference.

We use the term “high-resolution” broadly to include, for example, animage sensor that has a pixel spacing in one or both of two dimensionsthat is smaller than 5 µm, or 3 µm, or 1 µm, or sub-micron, for example.

We use the term “monolayer” broadly to include, for example, a volume ofa sample that has a thickness no greater than the thickness of aparticular type of unit in the sample, such as blood cells, so thatacross the monolayer two units cannot be stacked in the dimensiondefined by the thickness. In the case of a whole blood sample, thethickness of the monolayer could be in the range of 1 micrometer to 100micrometers.

Light 120 from a light source 122 illuminates the monolayer 100.Portions 124 of the light may pass through the sample monolayer and bereceived by photosensitive elements 126 in the array 128 of the imagesensor. Portions 130 of the light may be reflected or refracted bycomponents 131 of the monolayer and the reflected or refracted light maybe received by photosensitive elements in the array. Portions 132 of thelight may be transmitted through components of the monolayer and thetransmitted light may be received by photosensitive elements in thearray; portions of the light may be absorbed by components of themonolayer. As discussed later, the components of the monolayer caninclude countable units, chemical components, beads, and other elements.

The light source can be configured or controlled or both to provideilluminating light in one or more selected wavelength bands andcombinations of them. A wide variety of types of light sources andcombinations of them can be used, for example, LEDs, LED panels, organicLEDs, fluorescent panels, incandescent lamps, ambient illumination,arrays of monochrome LEDs, arrays of narrowband sources such as red,green, and blue LEDs or lasers, a miniaturized color display such as aliquid crystal or organic LED (OLED) display or an RGB laser colorprojector.

Using the light that originates at the light source and passes through,is reflected or refracted by, or is transmitted through the monolayer,the image sensor captures one or more images of the monolayer includingcountable units of various types (for example, blood cells) and chemicalcomponents that are detectable (either in their native condition or as aresult of being marked as discussed later). One or more of the capturedimages are processed by one or more processors or other image processingcomponents 113 to produce information 133 about the whole blood sampleincluding, for example, a CBC or a chemical analysis or both of thecountable units and chemical components. Among other things, theresulting information can include a count of red blood cells and theirhemoglobin content.

The CBC information can be generated by identifying and counting in thecaptured images the number of countable units of each type in thesample. Additional information about CBC techniques and about imagingusing contact image sensors can be found, for example, in U. S. Pat.Publications 2016/0041200, 2014/0152801, 2018/0284416, 2017/0293133,2016/0187235, and in U.S. Pats. 9,041,790, 9,720,217, 10,114,203,9,075,225, 9,518,920, 9,989,750, 9,910,254, 9,952,417, 10,107,997, allof which are incorporated here by reference.

As shown in FIG. 3 , a monolayer 140 of whole blood (such as the samemonolayer of whole blood used for the contact CBC technique) can be usedfor chemical analysis of various chemical components 142, 144 of thewhole blood. For this purpose, individual units of the different typesof chemical components of the whole blood monolayer sample can betreated as fillings of sandwiches 148 similar to the fluorescentsandwiches. However, in implementations of the sample analysistechnology described here, capture antibodies 150, 152 and detectionantibodies 154, 156 are attached to beads 158, 160, 162, 164 that neednot have fluorescent properties and are directly visible or otherwisedetectable using light that originates at the light source and passesthrough, is reflected or refracted by, or is transmitted through themonolayer or components of the monolayer. The resulting light isreceived by light-sensitive elements (e.g., pixel) 166 arrayed in theimage sensor 168. (Unlike fluorescence techniques, the light source isnot within the monolayer sample but is external to it.)

Using the received light (in some cases, the same received light usedfor the contact CBC technique), the image sensor captures one or moreimages of the monolayer sample. One or more processors 170 or otherimage processing devices process the one or more received images andapply a variety of techniques to identify the presence of and determinethe level (e.g., quantity, amount, volume, percentage) of each of thechemical components in the sample.

The beads 158, 160, 160, 162 to which the antibodies 150, 152 and 154,156 are attached need not have fluorescent properties. The beads canhave characteristics that are detectable, visible, or otherwisediscernible based on light from the light source that is reflected from,refracted by, or passes through them. We sometimes refer to such beadsas “direct indicator beads”. The direct indicator beads can take theform of what are sometimes call microbeads in reference to their smallsize. Microbeads have sizes typically in the range of 0.5 to 500micrometers.

We use the term “direct indicator beads” (or sometimes simply “beads”)broadly to include, for example, any tag, marker, or other indicatordevice or indicator characteristic that can be attached to or associatedwith a chemical component of a sample and is identifiable at a sensorusing received light that was incident on and reflected or refracted byor transmitted through the indicator device or characteristic. In somecases, direct indicator beads can take the form of small grains,particles, beads, spherules, or other elements, and combinations ofthem, and can be of a variety of shapes, sizes, materials, and colors.

To determine the presence of units of chemical components in the sample,the processor analyzes the images to detect directly discerniblecharacteristics of beads and complexes of two or more beads that arerevealed by light originating from the light source and reflected from,refracted by, or transmitted through the beads to the surface of theimage sensor.

We use the term “directly discernible characteristics” of beads andcomplexes of beads broadly to include, for example, any quality,attribute, or other trait that can be detected, determined, or derivedfrom light that originated at a light source and was reflected from,refracted by, or transmitted through the beads. Directly discerniblecharacteristics could include color, size, texture, birefringence, orshape, or combinations of them, for example.

We use the term “complexes of beads” broadly to include, for example,two or more beads that can be associated with one another because theyare attached to a unit in a sample, such as a molecule or other chemicalcomponent. Typically, the two or more beads of a complex are detectablein constant close proximity (e.g., touching) one to another. In somecases, the two or more beads of a complex are detectable because theyhave two or more predetermined different directly discerniblecharacteristics. For example, two beads of a complex may have twospecific different colors that are discernible by processing the imagesfrom the image sensor.

We use the word “attach” to include both direct and indirect attachment.For example, two particles, units, or other elements may be attacheddirectly (e.g., in contact and bound) to one another, or indirectly(e.g., attached to one another by an attachment unit bound to each ofthe two particles, units, or other elements).

We use the term “attachment unit” broadly to include, for example, anyantibodies (e.g., an antibody directed against acluster-of-differentiation cell surface antigen if the target unit is aspecific cell type), capsid proteins or other antigens from a pathogenicvirus (e.g., if the target unit is an antibody to the pathogenic virus,indicating prior exposure to the pathogenic virus), and other bindingmolecules and structures suitable for binding or attachment (e.g.,direct attachment) to a unit of a chemical component.

The sample analysis technology that we describe here can be applied in avariety of different modes.

In some examples of one such mode, which we sometimes call thecomplexed-beads mode, the chemical components remain dissolved orsuspended in the sample. A capture antibody and a detection antibody,each coupled to a separate direct indicator bead, bind simultaneously tothe two different locations on a given target molecule or other unit ofa target chemical component to form a complex of two beads (i.e., adoublet). [Because each direct indicator bead has more than one of itsparticular (capture or detection) antibody bound to its surface, a beadmay participate in more than one such complex simultaneously, forming atriplet or higher-order bead complex.]

By processing one or more images captured by the image sensor, it ispossible to identify those beads present in doublets or higher-ordercomplexes, and thus associated with the chemical component. Bydetermining the proportion of complexed beads to the total number ofbeads (complexed and singleton, that is, uncomplexed) identified in thesample, it is possible to determine the level or amount or quantity orconcentration of the target units (e.g., molecules) of the chemicalcomponent in the sample.

It is true that identified singleton beads are not necessarily beadsunbound to the target molecule, because in some cases only the captureantibody or the detection antibody, but not both, may have bound to thetarget molecule.

However, under constant incubation conditions and provided that theconcentrations of bead-coupled capture antibodies and bead-coupleddetection antibodies in the sample are constant and their ratio isknown, it is possible empirically to establish a “standard curve” thatrepresents the relationship between the bead complex index (that is, theproportion of complexed beads to total beads identified by the device)and the concentration of the target molecule.

This has been done experimentally for prolactin to generate the standardcurve shown in FIG. 4 . Using the standard curve, it is possible todetermine the otherwise unknown concentration of prolactin in a sampleby determining the bead complex index under identical incubationconditions.

In some implementations, the same beads can be used to mark both thecapture antibodies and the detection antibodies that will bind to givenunits of the chemical component. In some implementations, by usingcomplexes of beads having different directly discernible characteristicsfor capture antibodies and detection antibodies that are to be attachedto the units of different chemical components it is possible tomultiplex the process of detecting the presence and levels of thedifferent chemical components at the same time. Multiplexing can beachieved by using beads having different colors, sizes, shapes,textures, or other directly discernible characteristics.

As shown in FIG. 5 , in some implementations, the capture antibodies 200are irreversibly bound to a fixed surface 202, for example differenttypes of capture antibodies are bound as spots 206 in knowncorresponding locations in an array 204 on the fixed surface. In suchimplementations the capture antibodies need not have direct indicatorbeads attached to them, but the detection antibodies would have directindicator beads attached to them. The fixed surface could be the surface108 of the lid 110 that faces the surface 102 of the image sensor 104and defines a gap occupied by the monolayer 100 of the sample. When themonolayer of the sample is in the gap and in contact with the printedspots in the array of capture antibodies, respective chemical componentsin the sample will bind to respective capture antibodies, based on thetype of the chemical components, in positions defined by the locationsof the printed spots in the array, and can at the same time bind todetection antibodies coupled to direct indicator beads. Images capturedusing incident light that passes through the monolayer and is reflected,refracted or transmitted by the direct indicator beads can then beprocessed to identify and determine the amounts of different types ofchemical components based on the imaged locations of the beads attachedto the detection antibodies. This technique of chemical analysis can beused separately or in combination with the contact CBC techniquediscussed earlier.

In some implementations, a combination of the location-based chemicalanalysis technique and the in-solution or in-suspension (that is,non-location-based, complexed-beads mode) chemical analysis techniquecould be used.

In order to use these chemical analysis techniques in combination withthe CBC technique in a point-of-care setting, steps must be taken toimpart the bead-coupled antibodies to the sample before it is loadedonto the image sensor surface. One approach would be to pass the sampleof blood taken from the patient through a tube where dried bead-coupledantibodies are solubilized by the blood and allowed to bind with thetarget molecules. Then the prepared sample can be placed on the sensorsurface. Another approach would be to deposit the bead-coupledantibodies onto the surface 108 of the lid 110 (in some cases inaddition to bead-free capture antibodies irreversibly bound at specificlocations of the lid) so that they are solubilized when the lidencounters the blood sample in forming the monolayer.

In some implementations, the target unit of the chemical componentincludes at least one of an antigen, a hormone, a biomarker, a drug, aviral capsid, a pathogen-directed antibody (for example, avirus-directed antibody), an oligonucleotide, or another molecule, cell,or particle.

In some implementations, a bead is bound to an attachment unit, and theattachment unit is bound to the target unit of the chemical component.In the example of FIG. 3 , attachment units 150 and 154 are bound to afirst target unit 142 of a first chemical component, and attachmentunits 152 and 156 are bound to a second target unit 144 of a secondchemical component. Beads 158 and 160 are bound to attachment units 150and 154, respectively, to form a multi-bead complex 143 that includesbeads 158 and 160, attachment units 150 and 154, and target unit 142.Beads 162 and 164 are bound to attachment units 152 and 156,respectively, to form a multi-bead complex 145 that includes beads 162and 164, attachment units 152 and 156, and target unit 144. In someimplementations, it is the proximity of the beads 162 and 164 to oneanother or the constancy of the proximity or both that allows for theidentification of the multi-bead complex. The proximity can be measuredin terms of absolute distance, or proportion of the dimension of one ormore of the beads, for example.

The attachment units 150, 152, 154, 156 can be, but need not be,detection antibodies or capture antibodies. In addition to, oralternatively to, antibodies, the attachment units 150, 152, 154, 156may include a capsid protein or other antigen of a pathogenic virus. Theattachment units 150, 154 may be different from one another.

The target units 142 and 144 of the chemical component may include atleast one of an antigen, a hormone, a biomarker, a drug, a viral capsid,a pathogen-directed antibody (for example, a virus-directed antibody),an oligonucleotide, or another molecule, cell, or particle. Theattachment units 150, 152 (for example) may include proteins of a virus,the proteins binding to an antibody 142 of the virus.

In some implementations, the beads 158, 160 are different from oneanother in one or more characteristics such as size, color, shape,surface properties, translucency, weight, and combinations of them.

As shown in FIG. 6 , in some implementations, an attachment unit 306(for example, a capture unit, capture particle, or other captureelement) is bound at a known location 308 on a surface 310 (e.g., asurface of an image sensor, or a surface of a lid). The surface 310 mayinclude an array of known locations 312, each known locationcorresponding to a known type of attachment unit (not shown, except for306). The attachment unit 306 is bound to a target unit 304 of achemical component, which is bound to an attachment unit 302. Theattachment unit 302 is bound to a direct indicator bead 300. Asdescribed in reference to FIG. 5 , because the attachment unit 306 is ofa known type (e.g., a type known to bind to the target unit 304 of thechemical component) and in a known location, imaging of the directindicator bead 300 and determination of the known location 308 may beused to determine an amount or presence or both of the chemicalcomponent.

The attachment units 302 and 306 can be, but need not be, antibodies,and can include types of attachment units as described above inreference to FIG. 3 . The target unit 304 of the chemical component mayinclude at least one of an antigen, a hormone, a biomarker, a drug, aviral capsid, a pathogen-directed antibody (for example, avirus-directed antibody), an oligonucleotide, or another molecule, cell,or particle.

In some implementations, a capture bead includes an attachment unit. Insome implementations, a surface (for example, the surface 310) includesan attachment unit.

Various choices of the target units and attachment units may be used ina variety of applications, such as cytometry, in vitro diagnostics,environmental analysis, multiplex biochemical assays, serology, and geneexpression and combinations of them.

In an example of serology applied to a sample of blood from a patient,beads are bound to recombinant viral proteins of an infectious virus.The recombinant viral proteins bind to antibodies of the infectiousvirus within the sample. Complexes of two or more of the beadsassociated with an antibody of the infectious virus are identified orenumerated or both, and results of the identification or enumeration orboth (potentially in concert with the identification or enumeration orboth of single un-complexed beads) are used to determine a presence orlevel or both of the antibody of the infectious virus, as describedearlier. Based on the determined presence or level or both of theantibody of the infectious virus, past exposure by the patient to theinfectious virus can be identified. Samples besides blood may be used inaddition to or instead of blood.

Other implementations are also within the scope of the following claims.

1. An apparatus comprising: an image sensor having an array of light sensitive elements, and a first surface; and a computing device communicatively coupled to the image sensor, the computing device configured to: cause the image sensor to capture one or more images of a sample disposed on the first surface, wherein the sample includes a unit of a chemical component, the unit of the chemical component bound to a first attachment unit and a second attachment unit, wherein the first attachment unit is attached to a bead, and the second attachment unit is attached at a location on the first surface or on a second surface, and wherein the one or more images are captured based on light originating at a light source and reflected by, refracted by, or transmitted through the bead, receive, from the image sensor, data representative of the one or more images, identify the bead in at least one image of the one or more images of the sample, identify, in the at least one image of the one or more images of the sample, the location at which the second attachment unit is attached on the first surface or on the second surface based on the identification of the bead, and determine, based on a correspondence between the second attachment unit attached at the identified location and the chemical component, a presence or a level of the chemical component in the sample.
 2. The apparatus of claim 1, wherein the second attachment unit is attached to the first surface, and wherein the first surface comprises a sensor surface of the array of light sensitive elements.
 3. The apparatus of claim 1, comprising the second surface, wherein the second surface comprises a top surface facing a sensor surface of the array of light sensitive elements, and wherein the second attachment unit is attached to the second surface.
 4. The apparatus of claim 3, wherein the top surface is arranged to form a monolayer of the sample between the top surface and the sensor surface of the array of light sensitive elements.
 5. The apparatus of claim 1, wherein the first attachment unit and the second attachment unit comprise antibodies.
 6. The apparatus of claim 1, wherein the first attachment unit and the second attachment unit comprise antigens from a pathogen, and wherein the unit of the chemical component comprises an antibody to the pathogen.
 7. The apparatus of claim 6, wherein the pathogen comprises a virus.
 8. The apparatus of claim 1, wherein the second attachment unit is printed on the first surface or the second surface.
 9. The apparatus of claim 1, wherein a plurality of attachment units, including the second attachment unit, are attached at respective locations on the first surface or the second surface, the respective locations forming an array.
 10. The apparatus of claim 1, wherein the sample comprises whole blood of a human or animal.
 11. An apparatus comprising: an image sensor having an array of light sensitive elements, and a surface; and a computing device communicatively coupled to the image sensor, the computing device configured to: cause the image sensor to use the array of light sensitive elements to capture one or more images of a sample disposed on the surface, wherein the sample includes a plurality of multi-bead complexes, each multi-bead complex of the plurality of multi-bead complexes comprising two or more respective beads attached to a respective unit of a chemical component, and wherein the one or more images are captured based on light originating at a light source and reflected by, refracted by, or transmitted through the two or more respective beads of each multi-bead complex, receive, from the image sensor, data representative of the one or more images, measure, in at least one image of the one or more images of the sample, a proximity to one another of the two or more respective beads of at least two different multi-bead complexes of the plurality of multi-bead complexes, and identify, in the at least one image, two or more separate multi-bead complexes based on the measured proximity to one another of the two or more respective beads of the at least two different multi-bead complexes.
 12. The apparatus of claim 11, wherein the computing device is configured to identify a presence or a level of the chemical component in the sample, based on the identification of the two or more separate multi-bead complexes.
 13. The apparatus of claim 11, wherein the computing device is configured to enumerate a number of the plurality of multi-bead complexes in the sample.
 14. The apparatus of claim 11, wherein the sample comprises attachment units, and wherein, for each multi-bead complex comprising two or more respective beads attached to the respective unit of the chemical component, two or more respective attachment units are bound to the respective unit of the chemical component, and each attachment unit of the two or more respective attachment units is bound to at least one bead of the two or more respective beads, such that each multi-bead complex of the plurality of multi-bead complexes comprises the two or more respective beads, the two or more respective attachment units, and the respective unit of the chemical component.
 15. The apparatus of claim 14, wherein the two or more respective attachment units of each multi-bead complex of the plurality of multi-bead complexes comprise antibodies.
 16. The apparatus of claim 14, wherein the two or more respective attachment units of each multi-bead complex of the plurality of multi-bead complexes comprise antigens from a pathogen, and wherein the respective unit of the chemical component of each multi-bead complex of the plurality of multi-bead complexes comprises an antibody to the pathogen.
 17. The apparatus of claim 16, wherein the pathogen comprises a virus.
 18. The apparatus of claim 14, wherein at least two attachment units of the two or more respective attachment units of each multi-bead complex of the plurality of multi-bead complexes are different from one another.
 19. The apparatus of claim 14, wherein the two or more respective attachment units of each multi-bead complex of the plurality of multi-bead complexes are configured to bind at different locations at the respective unit of the chemical component of the multi-bead complex.
 20. The apparatus of claim 11, wherein at least two beads of the two or more respective beads of a first multi-bead complex of the plurality of multi-bead complexes differ in at least one of appearance, reflective characteristics, refractive characteristics, or transmissive characteristics, for the light originating at the light source. 